PROJECT SUMMARY The CRISPRi/a core will support research of Projects 1, 2, and 3 by enabling knockdown and overexpression of endogenous genes in human iPSC-derived neurons. The CRISPRi/a technology, which we co- developed, enables highly specific, inducible and reversible control of gene expression in mammalian cells. We use a catalytically inactive version of the bacterial Cas9 protein (dCas9) to recruit transcriptional repressors (for CRISPRi) or transcriptional activators (for CRISPRa) to endogenous genes, as directed by single guide RNAs (sgRNAs). We have established the use of this technology in two modes: to investigate the function of individual genes of interest (reverse genetics), and to conduct genome-wide screens to uncover genes relevant for a biological process of interest (forward genetics). We will support the research of Projects 1, 2, and 3 by enabling CRISPRi/a-based forward and reverse genetics in human iPSC-derived neurons. First, we will generate and validate stable CRISPRi and CRISPRa cell lines from the isogenic human iPSCs expressing wild-type tau or V337M tau that are used by Projects 1, 2, and 3. Then, we will generate and validate sgRNAs targeting axon initial segment (AIS) proteins to enable the investigation of their effects on plasticity and excitability of V337M tau neurons (for Project 1), sgRNAs targeting key autophagy pathways to address the question if modulation of these pathways can restore neuronal excitability of V337M tau neurons (for Projects 1, 2, and 3), and sgRNAs targeting proteins selectively interacting with V337M tau that could underlie the abnormality in neuronal activity and autophagy pathways induced by pathogenic seeding (for Projects 1 and 3). We will also conduct two genome-wide CRISPRi screens. First, we will aim to identify cellular pathways controlling tau uptake, which will then be further characterized by Project 2. Second, we will aim to identify cellular pathways controlling templates tau aggregation. These forward genetics approaches will complement the hypothesis-driven reverse-genetics approaches and provide an unbiased survey of relevant cellular pathways. We will work with the Data core to integrate our datasets with those generated by the MS core, with the goal to identify convergent results from the proteomic and genetic approaches, and to work with the Human core to validate the relevance of our cell-based findings in human patients.